Regulation of Respiratory and Ligninolytic Enzyme Activity of Lentinula Edodes by Selenium

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Advances in Microbiology, 2013, 3, 31-36 Published Online December 2013 (http://www.scirp.org/journal/aim) http://dx.doi.org/10.4236/aim.2013.38A006

Regulation of Respiratory and Ligninolytic Enzyme Activity of Lentinula edodes by Selenium

Regiane Gonçalves Feitosa Leal Nunes1, Jose Maria Rodrigues da Luz2, Elizabete Fantuzzi3, Maria Catarina Megumi Kasuya2, Maria Cristina Dantas Vanetti2* 1Instituto Federal de Educação, Ciência e Tecnologia do Piauí, Campus Teresina Central, Centro, Teresina, Brazil 2Department of Microbiology, Federal University of Viçosa, Viçosa, Brazil 3Federal University of Alegre, Alegre, Brazil Email: [email protected], [email protected], *[email protected]

Received August 9, 2013; revised September 9, 2013; accepted September 16, 2013

Copyright © 2013 Regiane Gonçalves Feitosa Leal Nunes et al. This is an open access article distributed under the Creative Com- mons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT The production of mushrooms enriched with essential elements, e.g. selenium, for human health is an interesting strategy to improve the functional foods supply. The selenium is an essential mineral and makes part of structure of enzymes involved in the oxidative metabolism. However, the selenium effect on the activity of respiratory and lignocellulolytic enzymes has not been considered. The understanding of this effect is important to determine the selenium concentration that increases the mushroom productivity and the degradation rate of the substrate. In this study, it was observed reduction of the respiratory activity of Lentinula edodes (Berk.) Pegler, the shiitake mushroom, in function of the increasing of the sodium selenite concentration in the substrate (p < 0.05). Selenium did not inhibit the activity of the hydrolytic enzymes (cellulase and xylanase), but it increased the activity of the oxidative enzyme (laccase). Respiratory activity of L. edodes has a negative correlation with sodium selenite concentration added in substrate. Thus is important to define the ideal dose of selenium to be added to the substrate for increasing lignocellulosic residues degradation and, consequently, guarantee a higher production of Se-enriched mushrooms.

Keywords: Shiitake; Laccase; Se-Enriched Mushrooms; Eucalypt Sawdust; Lignocellulosic Residues

1. Introduction mushrooms are a versatile food, low in energy and fat but containing considerable amounts of protein, dietary fi- Agro-industrial activities have resulted in the accumula- bers, vitamins and mineral. They have effective mecha- tion of lignocellulosic residues and the fungi have an im- nisms for absorption and accumulation of some elements, portant function in the decomposition of this organic such potassium (K), iron (Fe), copper (Cu), zinc (Zn), matter. The use of these residues as carbon and energy selenium (Se), lithium and other metalloids [9-12], and source depends on the capacity of the fungus to produce depending on the fungal growth conditions, these mush- and excrete lignocellulolytic enzymes [1]. rooms can be enriched with minerals which present low Lentinula edodes (shiitake) is a saprotrophic fungus availability in the soil or in the foods [9,12]. that degrades lignocellulosic residues and produces lig- L. edodes has been showed as an alternative for pro- nocellulolytic enzymes as laccase, manganese peroxidase, duction of Se-enriched food [11,13]. However, the pro- cellulase and xylanase [2-6]. Besides of the lignocellu- ductivity of mushrooms can be influenced by the activity lolytic enzymes production of industrial interest, L. edo- of respiratory and lignocellulolytic enzymes, which has des is capable of converting lignocellulosic residues, e.g. not been investigated. In Pleurotus ostreatus, high level eucalypt logs and sawdust, coffee husks, and corncobs, in of Se in the substrate, decreases the productivity and al- mushrooms that can be used in the human and animal ters the morphology of the mushrooms [12]. feeding [1,7,8]. Selenium is a metalloid naturally found in the soil and In addition to be considered as delicacies, the edible part of the structure of enzymes like glutathione per- *Corresponding author. oxidase, deiodinase, thioredoxin redutase and selenopro-

Open Access AiM 32 R. G. F. L. NUNES ET AL. tein P, which were involved in the oxidative metabolism sodium acetate buffer (0.1 mol·L−1 and pH 4.8) and agi- [14,15]. Furthermore, the selenate and selenite reduction tated at 150 rpm for 30 mim [3,18]. Next, the suspension to elementary selenium can be catalyzed by microbial was filtered in Millipore membranes (Whatman 1). enzymes [16]. Laccase activity was determined by oxidation of the Thus, the objective of this study was to evaluate the 2,2’-azino-bis-3-ethil benzothiazole-6-sulfonic acid (ABTS) effect of Se in the respiratory and enzymatic activity of L. at 37˚C [19]. The enzymatic reaction was prepared with edodes growing in lignocellulosic residue. 0.6 mL of sodium acetate buffer (0.1 mol·L−1 and pH 5.0), 0.3 mL of EE and 0.1 mL of ABTS (1 mol·L−1). This 2. Material and Methods reaction was incubated in a water bath at 37˚C for 10 min and, subsequently, absorbance was determined at 420 nm. 2.1. Microorganism One unit of laccase/enzyme activity was defined as the In this study, UFV 11, UFV 16 and UFV 53 L. edodes amount of enzymes required for oxidation of 1 µmol of (Berk.) Pegler isolates from the collection of the De- ABTS per min at 37˚C. partment of Microbiology of the Federal University of Cellulase and xylanase activities were determined by Viçosa (MG, Brazil) were used. These isolates were ob- the amount of released reducing sugars from filter paper tained from basidiocarps of commercial mushrooms that (Whatman 1) and xylan, respectively [3,20,21]. The filter were produced in Moji das Cruzes, São Paulo, Brazil. paper and xylan were used as enzymatic substrate. One The stock cultures were maintained on potato dextrose units of these hydrolases activity was defined as the agar (PDA, Merck). Mycelium of each culture was amount of enzymes catalyzing the release of 1 mmol·L−1 grown at 25˚C on the surface PDA agar in Petri dishes. of glucose or xylose per min at 50˚C. Reducing sugars were determined by dinitrosalicylic 2.2. Growth Conditions and Analyses of acid method (99.5% dinitrosalicylic acid, 0.4% phenol Respiratory Activity and 0.14% sodium metabisulfite) [3,22]. Soluble protein was determined by the colorimetric Three L. edodes isolates were cultivated in 20 g of sub- method described by Bradford [23] and used for calcula- strate based on eucalypt sawdust (78% eucalypt sawdust, tion of enzymes specific activity. 20% rice bran, 0.4% CaCO3 and 1.6% CaSO4) with addition of 0.32, 0.64, 0.96 or 1.28 mmol·L−1 of sodium se- 2.4. Statistical Analysis lenite (Na2SeO3) [11]. Tap water was added to reach 65% moisture content. Factorial delineation was used with three fungal isolates The substrates were placed in glass flasks (150 mL), (UFV 11, UFV 16 and UFV 53), five Se concentrations autoclaved at 121˚C for 20 min and, after cooling, each (0, 0.32, 0.64, 0.96 and 1.28 mmol·L−1), three incubation flask was inoculated with one agar disc (8 mm diameter) time (7, 14 and 21) and three replicates. The data were containing fungal mycelium grown in PDA for 15 d. subjected to analysis of variance and regression (p < 0.05) These flasks were incubated at 25˚C for 21 d. The control using Saeg software (version 9.1, Federal University of treatment was the eucalypt sawdust without addition of Viçosa). Na2SeO3 and incubated in the same experimental conditions. 3. Results For determine respiratory activity the flasks were cou- The respiratory activity of L. edodes was different among pled to the respirometer of continuous flow with detector isolates (p < 0.05) and decreased in function of sodium of infrared (TR-RM8 Respirometer Multiplexer—Sable selenite concentration in substrate. In isolate UFV53, this systems). CO production measurement was carried out 2 reduction was higher than 40% and, for each isolate a every seven hours [17] and bottles remained coupled to specific regression model was obtained (p < 0.05, Figure the respirometer for seven days. 1). These models are important to determine the sodium selenite dose that should be added in the substrate for a 2.3. Enzymatic Assay and Protein Quantification better fungal growth. Every seven incubation days substrate samples with fun- Se enhanced the laccase specific activity (p < 0.05) gal growth were collected for analyses of laccase (EC when compared with the treatment without addition of 1.10.3.2), cellulase (EC 3.2.1.4), xylanase (EC 3.2.1.8/ this element (Figure 2). The activity of this enzyme was EC 3.2.1.32/EC 3.2.1.136) specific activities and for different among the isolates (p < 0.05) with higher value soluble protein determination. obtained by UFV11, at of 0.96 and 1.28 mmol·L−1 con- For protein extraction and preparation of enzyme ex- centrations (Figure 2). tracts (EE), 3 g of the substrate colonized by the fungi Cellulase specific activity was low and presented sig- were put in Erlenmeyer (125 mL) containing 10 mL of nificant difference (p < 0.05) between sodium selenite

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Se on the growth of L. edodes, Nunes et al. [11] sug- gested the addition of up to 1.28 mmol·L−1 of sodium selenite in the water cold shock that is used to induce fructification of mushroom after mycelial growth. The inhibitory effect maybe avoided due to the smallest time of exposition of the fungus to the high doses of sodium selenite. No correlation between the reduction of the respiratory activity with increase of the sodium selenite concentration (Figure 1) and lignocellulolytic enzymes activity was observed (Figure 2). This fact can be due to factors 0 0.32 0.64 0.96 1.28 such as 1) greater synthesis of extracellular enzymes to Sodium selenite (mmol·L−1) keep fungal growth under conditions of stress, 2) the presence of the selenium in the substrate do not inhibiting the lignocellulolytic enzymes activity or 3) this element have an activator effect on the activity of these en- Figure 1. Lentinula edodes respiratory activity, UFV11, zymes. The last two factors seem to be most probable, UFV16 and UFV53 isolates, cultivated in the eucalypt saw- because enzymatic activity was greater in the presence of dust containing or not different sodium selenite doses, dur- the selenium (Figure 2). Although reports have not been * ing 21 days at 25˚C. Significant regression model (p < 0.05). found in the literature on the effect of the Se in the microbial enzyme activity, several studies have showed that concentrations added to the substrate and also between the laccase activity is inhibited in presence of certain the analyzed strains (Figure 2). The highest activities of metals like iron, mercury and silver [28], Cd, lead (Pb) this enzyme were observed in the cultivation of isolates −1 and mercury [29]. Cadmium, Cu, Pb, manganese (Mn) UFV11 and UFV16 in substrate with 1.28 mmol·L of and Zn also affected cellulase and xylanase activity of P. sodium selenite (Figure 2). ostreatus cultivated in lignocellulosic residues [30,31]. The xylanase specific activity was also low, varied in Nevertheless, the presence of minerals such as Cu, Mn function of the addition of sodium selenite in substrate and Zn increased ligninases activity of different Basi- and the incubation time (Figure 2) and, no difference diomycetes [3,30,32]. Hence, the selenium might have among the isolates of L. edodes was observed (p < 0.05). been an activator of laccase in this study. A high specific activity of the laccase was also ob- 4. Discussion served during P. ostreatus growth in different lignocel- The reduction of the respiratory activity (p < 0.05) shows lulosic residues [18] and L. edodes in eucalypt bark [3]. that Se in concentration greater than 0.96 mmol·L−1 can In contrast with the present study, Cavallazzi, Brito, be toxic to the fungus (Figure 1). This toxicity had also Oliveira, Villas-Bôas and Kasuya [3] showed that of this been observed by Nunes [24] once that the mycelial enzyme declines with incubation time. Ohga and Royse growth of L. edodes in Petri dish, containing PDA or [7] noticed that the laccase activity of L. edodes was eucalypt sawdust, decreased as the sodium selenite dose higher during the mycelial growth, decreasing quickly in in the culture medium was increased. According to this the beginning of the fructification period. According to author, the inhibitory effect of mycelial growth was ob- the last authors [7] this behavior is due to the fact that, served regardless the composition of the cultivation me- the expression of the laccase genes, measured as mRNA dium and, in the concentrations greater than 1 mmol·L−1 levels, is maximum during the substrate colonization and the colony diameter was halved. The Se concentration decrease during fructification. So, the activity of this higher than 12.8 mg·kg−1 also decreased the mycelial enzyme can also be used for monitored of the fungal de- growth and altered the shape of the P. ostreatus mush- velopment in a specific substrates or in substrate en- room [12]. The toxicity of high doses of other metals riched with mineral or still to determine the inhibitory such as cobalt, cadmium (Cd) and magnesium have also effect of a element on the fungal growth. been shown in the growth of Psathyrella atroumbonata The highest reduction of the respiratory activity and [25] and Paxillus involutus [26]. Furthermore, the fungus lowest laccase activity observed in UFV53 may indicate stress induced by toxicity of certain metals depends on the greatest sensibility of this isolate to the Se (Figures 1 nutrients availability, as observed in Trichoderma viride and 2) or the physiological characteristic of this isolate, and Rhizopus arrhizus, cultivated in culture medium with because, it presented high manganese peroxidase activity low nutritional level or supplemented with Cd, Cu and and low laccase activity in eucalypt bark without addi- Zn [27]. To avoid the inhibitory effect of high doses of tion of Se [3,18]. Therefore, may have not occurred an

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Figure 2. Specific activity of lignocellulolytic enzymes (laccase, cellulase and xylanase) of Lentinula edodes, UFV11, UFV16 and UFV53 isolates, cultivated in eucalypt sawdust containing or not different sodium selenite doses, during 21 days at 25˚C. increasing in the expression of extracellular laccase to tions used, once; the biosynthetic potential of Basidio- compensate the reduction of respiratory activity. And, in mycetes is highly dependent of the growing conditions this case, Se also is not inhibiting the enzymatic activity, [5]. Thus, the low cellulase activity (Figure 2) can be in unlike, it can have activated the laccase activity (Figure function of the chemical composition of eucalypt saw- 2). dust, which presents higher lignin levels than cellulose Although low cellulase and xylanase activities were and hemicelluloses and, not necessarily, due to the addi- observed, selenium did not inhibit the activity of these tion of sodium selenite to the substrate. hydrolases that are responsible for cellulose and hemi- The low xylanase activity and the reduction of this ac- cellulose degradation (Figure 2). This low cellulase activity along of incubation time can also be related to the tivity and the variation of the activity of this enzyme substrate composition (Figure 2) because, the main fac- among the isolates of L. edodes (Figure 2) were also tors to an efficient production of xylanase by microor- shown by Cavallazzi, Brito, Oliveira, Villas-Bôas and ganisms are the appropriate choice of the substrate and Kasuya [3] and Philippoussis et al. [5]. According to the optimization of the composition of the culture me- Leatham [2] L edodes is a moderately cellulolytic fungus, dium [33]. with low exo-1,4-D-glucanase activity. In contrast, Silva, Cellulase and xylanase activities were higher at days Machuca and Milagres [5] observed high cellulase activ- seven and fourteen of cultivation, while laccase activity ity of L. edodes that was quite uniform among the evalu- was higher at day twenty-one (Figure 2). Considering ated isolates. The difference between this study and that that during white rot fungus growth in lignocellulosic carried out by Silva, Machuca and Milagres [5] may be substrate, the laccase acts firstly in the complex polymers due to the substrate composition and cultivation condi- degradation releasing cellulose and hemicelluloses im-

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